Experimental and theoretical evidence supporting the role of Gly363 in blood coagulation factor IXa (Gly193 in chymotrypsin) for proper activation of the proenzyme.
Factor IX is the zymogen of the serine protease factor IXa involved in blood coagulation. In addition to a catalytic domain homologous to the chymotrypsin family, it has Ca2+, phospholipid, and factor VIIIa binding regions needed for full biologic activity. We isolated a nonfunctional factor IX protein designated factor IXEagle Rock (IXER) from a patient with hemophilia B. The variant protein is indistinguishable from normal factor IX (IXN) in its migration on sodium dodecyl sulfate-gel electrophoresis, isoelectric point in urea, carbohydrate content and distribution, number of gamma-carboxyglutamic acid residues, and beta-OH aspartic acid content, and in its binding to an anti-IXN monoclonal antibody which has been shown previously to inhibit the interaction of factor VIIIa with factor IXaN. Further, IXER is cleaved to yield a factor IXa-like molecule by factor XIa/Ca2+ at a rate similar to that observed for IXN. However, in contrast to IXaN, IXaER does not bind to antithrombin-III (specific inhibitor of IXaN) and does not catalyze the activation of factor X (substrate) to factor Xa. To identify the mutation in IXER, all eight exons of IXN and IXER gene were amplified by the polymerase chain reaction technique and cloned. A single point mutation (G----T) which results in the replacement of Val for Gly363 in the catalytic domain of IXER was identified. Gly363 in factor IXa corresponds to the universally conserved Gly193 in the active site sequence of the chymotrypsin serine protease family. X-ray crystallographic data in the literature demonstrate a critical role of this Gly in stabilizing the active conformation of chymotrypsin/trypsin in two major ways: 1) in the formation of the substrate binding site; and 2) in the development of the oxyanion hole. Our computer structural data support a concept that the Gly363----Val change prevents the development of the active site conformation in factor IXa such that the substrate binding site and the oxyanion hole are not formed in the mutated enzyme.
Bajaj
S P
SP
Department of Medicine, St. Louis University School of Medicine, Missouri 63104.
Spitzer
S G
SG
Welsh
W J
WJ
Warn-Cramer
B J
BJ
Kasper
C K
CK
Birktoft
J J
JJ
D003196:Computer Graphics; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism; D015949:Factor IXa / Q000378:metabolism*; D005170:Factor X / Q000378:metabolism*; D005998:Glycine; D006467:Hemophilia A / Q000097:blood; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D016133:Polymerase Chain Reaction; D011487:Protein Conformation
D004792:Enzyme Precursors; D005170:Factor X; D015949:Factor IXa; D005998:Glycine